The invention relates to a vertebrate intestinal protein which absorbs cholesterol. The protein is identified by means of high-affinity crosslinking compounds. The invention further relates to the use of the protein for carrying out a method for identifying a compound which inhibits cholesterol transport in the intestine.
In humans, on average about 50% of the cholesterol is present in the lumen of the intestine. The intraluminal cholesterol originates mainly from the diet and from the bile. About 2 g of cholesterol a day is discharged from the bile. The intestinal cholesterol absorption depends greatly on the presence of bile salts. Thus the effect of administration of inhibitors of the reuptake of bile salts or of bile salt sequestrants is to inhibit intestinal cholesterol absorption.
Inhibition of intestinal cholesterol absorption is an important aim of the treatment of lipid disorders, atherosclerosis and cardiovascular disorders. The prevailing opinion amongst experts is that intestinal cholesterol absorption takes place by physicochemical diffusion.
A number of observations in connection with cholesterol transport which indicate that a protein is involved are known. Intestinal cholesterol absorption is subject to great individual variability. Biochemical data from in vitro experiments indicate that proteins are involved in cholesterol exchange between small unilamellar vesicles and the brush border vesicles of the intestine. It was possible to observe large differences in the intestinal absorption of plant sterols such as xcex2-sitosterol and campesterol, which differ only in a methyl group (xcex2-sitosterol) and an ethyl group (campesterol). In humans, xcex2-sitosterol showed inter alia an inhibition of cholesterol absorption. There are two highly active classes of compounds which inhibit intestinal cholesterol absorption on luminal administration. The compounds are, on the one hand, compounds derived from saponin, such as tiqueside and pamaqueside, and on the other hand certain derivatives of 2-azetidinones. Derivatives of 2-azetidinones as inhibitors of cholesterol absorption are described in Clader et al., J. Med. Chem. 39, 3684-3693, 1996. For the purposes of this invention, absorption is intended to mean attachment of a substance to a protein and transport of this substance with the aid of this protein.
Several proteins have to date been thought to be associated with cholesterol absorption. However, as yet no protein unambiguously involved in cholesterol absorption in the intestine has been described. This leads to a number of disadvantages in the search for a rational procedure for identifying specific inhibitors of intestinal cholesterol absorption. This is of particular importance also because inhibitors of intestinal cholesterol absorption are especially suitable for the treatment of all types of lipid disorders, atherosclerosis and cardiovascular disorders. Cholesterol is distributed uniformly in all cells both in the lipid bilayer of the membranes and in lipid vesicles. Transport measurements with vesicles or cells are therefore very complex and subject to interference.
It is therefore an object of the present invention to provide a protein which is involved in intestinal cholesterol absorption.
Accordingly, in one embodiment, the instant invention provides an isolated cholesterol-absorbing protein from intestinal tissue of a mammalian organism which has been obtained by a process comprising: a) detecting said protein by contacting intestinal cells or parts of intestinal cells of a mammalian organism with a radiolabeled compound which acts as an inhibitor of intestinal cholesterol absorption, wherein said compound comprises a photolabile group; and b) isolating said protein.
In another embodiment, the instant invention also provides a pharmaceutical composition comprising a cholesterol-absorbing protein and a pharmaceutically acceptable excipient.
In another embodiment, the instant invention also provides a method of inhibiting cholesterol uptake comprising administering to a patient in need thereof an effective amount of a cholesterol-absorbing protein.
In another embodiment, the instant invention also provides a method of treating a disorder of cholesterol uptake or cholesterol excretion, lipid disorders, atherosclerosis, or cardiovascular disorders comprising administering to a patient in need thereof an effective amount of a cholesterol-absorbing protein.
In another embodiment, the instant invention also provides a method for identifying a compound which inhibits intestinal cholesterol absorption, comprising: a) contacting a cholesterol-absorbing protein from an intestinal tissue of a mammalian organism with a test compound; and b) detecting binding of said compound to said protein, whereby compounds that bind to said protein are identified as inhibitors of intestinal cholesterol absorption. Preferably, the method comprises contacting intestinal cells or parts of intestinal cells comprising a cholesterol-absorbing protein with a test compound.
In another embodiment, the instant invention provides compounds that are inhibitors of cholesterol-absorbing proteins.
In another embodiment, the instant invention provides a pharmaceutical composition comprising an effective amount of one or more compounds of the instant invention and a pharmaceutically acceptable excipient.
In another embodiment, the instant invention provides a method of inhibiting cholesterol uptake comprising administering to a patient in need thereof an effective amount of one or more compounds of the instant invention.
In another embodiment, the instant invention provides a method of treating a disorder of cholesterol uptake or cholesterol excretion, lipid disorders, atherosclerosis, or cardiovascular disorders comprising administering to a patient in need thereof an effective amount of one or more compounds of the instant invention.
In another embodiment, the instant invention provides a method for detecting the presence of a cholesterol-absorbing protein, comprising: a) contacting a protein with a compound according to the invention that binds the protein; b) detecting binding of said compound to said protein, whereby a protein that binds to said compound is identified as a cholesterol-absorbing protein.
In another embodiment, the instant invention provides a diagnostic kit for an assay for detecting the presence of a cholesterol-absorbing protein comprising one or more compounds of the instant invention.
In another embodiment, the instant invention provides a chemical conjugate comprising a polymer covalently linked to an inhibitor of intestinal cholesterol-absorption. Preferably, the polymer is covalently linked to a compound of the instant invention, further comprising a cholesterol-absorbing protein connected thereto.
In another embodiment, the instant invention provides a method for obtaining a cholesterol-absorbing protein, comprising: a) contacting parts of a disrupted cell comprising a cholesterol-absorbing protein with a chemical conjugate according to the instant invention to form a conjugate comprising a polymer covalently linked to an inhibitor of intestinal cholesterol-absorption and a cholesterol-absorbing protein connected thereto; and b) separating said cholesterol-absorbing protein from said conjugate to obtain said cholesterol-absorbing protein.
In another embodiment, the instant invention provides precursors of inhibitors of cholesterol-absorbing protein.
Additional objects, features and advantages of the invention will be set forth in the description which follows, and in part, will be obvious from the description, or may be learned by practice of the invention. The objects, features and advantages of the invention may be realized and obtained by means of the instrumentalities and combination particularly pointed out in the appended claims.